Phthalate dioxygenase reductase (PDR) is a prototypical iron-sulfur flavoprotein (36 kilodaltons) that utilizes flavin mononucleotide (FMN) to mediate electron transfer from the two-electron donor, reduced nicotinamide adenine nucleotide (NADH), to the one-electron acceptor, [2Fe-2S]. The crystal structure of oxidized PDR from Pseudomonas cepacia has been analyzed at 2.0 angstrom resolution resolution; reduced PDR and pyridine nucleotide complexes have been analyzed at 2.7 angstrom resolution. NADH, FMN, and the [2Fe-2S] cluster, bound to distinct domains, are brought together near a central cleft in the molecule, with only 4.9 angstroms separating the flavin 8-methyl and a cysteine sulfur ligated to iron. The domains that bind FMN and [2Fe-2S] are packed so that the flavin ring and the plane of the [2Fe-2S] core are approximately perpendicular. The [2Fe-2S] group is bound by four cysteines in a site resembling that in plant ferredoxins, but its redox potential (-174 millivolts at pH 7.0) is much higher than the potentials of plant ferredoxins. Structural and sequence similarities assign PDR to a distinct family of flavoprotein reductases, all related to ferredoxin NADP(+)-reductase.

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.

The purpose of this study is to characterize the interactions among P450 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. Previously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [Cawley et al. (1995) Biochemistry 34, 1244-1247]. The goal of the current study is to distinguish between two models to explain these interactions: one model where substrate increases the affinity of one P450 enzyme for the reductase, and another model where substrate increases the affinity of one P450 for the reductase through the formation of a 1A2-2B4 complex. According to this model, the 1A2 moiety of 1A2-2B4 forms a high-affinity complex with reductase. Reductase, 1A2, and 2B4 were reconstituted with dilauroylphosphatidylcholine, and the effect of reductase concentration on 7-pentoxyresorufin-O-dealkylation was examined with 2B4-reductase and 1A2-reductase binary systems, and in ternary systems containing different 2B4:1A2 ratios. At subsaturating [reductase], there was a dramatic inhibition of the 2B4-dependent activity in the ternary system as compared with the binary systems. These results are consistent with the formation of a ternary (reductase-1A2-2B4) complex where the reductase is bound specifically to 1A2. At higher reductase concentrations where the reductase-binding sites on 1A2 become saturated, the results are consistent with the formation of a quaternary complex in which reductase binds to both P450 enzymes (reductase-1A2-2B4-reductase). Analogous experiments using the 1A2-preferred substrate 7-ethoxyresorufin showed a stimulation of 7-ethoxyresorufin-O-deethylation in the mixed reconstituted system, demonstrating that the high-affinity 2B4-1A2-reductase complex was functionally active and not merely an inhibitory complex.

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

The goal of these studies was to demonstrate that one P450 isozyme can influence the function of another isozyme when combined in a reconstituted system. Benzphetamine and 7-pentoxyresorufin were both shown to be preferred substrates for P450 2B4 (LM2) as compared to P450 1A2 (LM4). However, these substrates exhibited different characteristics when examined in reconstituted systems containing reductase and both P450 isozymes. Whereas benzphetamine demethylation showed a small increase in catalytic activity when both P450 1A2 and 2B4 were present over the activities obtained in simple reconstituted systems, 7-pentoxyresorufin O-dealkylation (PROD) was dramatically inhibited when both isozymes were present. These results indicate that the functional interactions between P450s in complex reconstituted systems are dependent on the substrate present. Inhibition of PROD was also dependent on reductase levels, with the inhibitory effect being more pronounced at subsaturating reductase. Finally, these protein-protein interactions were shown to be dependent on the reductase concentration in the reconstituted system rather the P450 concentration, supporting the view that P450 1A2 is inhibiting the reaction by competing with P450 2B4 for reductase molecules.

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Phthalate dioxygenase reductase (PDR) is an electron transferase that contains FMN, which accepts a hydride from NADH, and a [2Fe-2S] center, which transfers electrons to phthalate dioxygenase. The reduction of PDR by NADH has been studied by stopped-flow spectroscopy. Data from studies using both portio- and deuterio-NADH were analyzed by nonlinear curve fitting and numerical simulation techniques. The results of these analyses indicate that the reductive half-reaction of PDR consists of five distinct kinetic phases: (a) NADH binds to form a primary Michaelis complex (MC-1) (Kd = 50 microM). (b) The enzyme undergoes a structural change (116 +/- 5 s-1) resulting in a charge-transfer complex (CT-1). (c) The next phase in the reaction shows a deuterium isotope effect of 7.0 when (4R)-[2H]NADH (NADD) is substituted for NADH, identifying this step as the one involving hydride transfer. The rate of hydride transfer from NADH to FMN is 70 s-1, and this process results in a charge-transfer intermediate between the flavin hydroquinone anion and NAD (CT). (d) Internal electron transfer from the flavin to the iron-sulfur center, which is only 35 +/- 4 s-1, then results in an intermediate consisting of a reduced [2Fe-2S] center and a neutral flavin semiquinone (SQ). It is surprising that this rate is so slow, since the shortest interatomic distance between these centers is only 4.7 A [Correll, C. C., et al. (1992) Science 258, 1604-1610]. The 2-electron-reduced form of PDR (SQ in Figure 1) binds weakly to the reaction product, NAD (Kd = 3.7 mM), but forms a tight complex with NADH (Kd = 10 microM). (e) Two molecules of the reduced iron-sulfur flavin semiquinone (SQ) form of PDR then undergo a relatively slow second-order disproportionation reaction, resulting in one molecule of 3-electron-reduced PDR and one molecule of 1-electron-reduced PDR. The latter reacts rapidly with excess NADH to form a 3-electron-reduced PDR.